An estimated 60-70 percent of human breast cancers are associated with sex hormone exposure. Approximately 60 percent of all breast cancer patients have hormone-dependent breast cancer, which contains estrogen receptors and requires estrogen for tumor growth. The possible biochemical roles of estrogens in the development of breast cancers remains to be fully elucidated. This research focuses on examination of the role of steroid hormones and estrogen biotransformations in breast cancer etiology. Our hypothesis is that alterations in the breast cancer tissue microenvironment can influence the extent of estrogen biosynthesis and metabolism, result in altered levels of hormonally active estrogens and their metabolites, and therefore influence breast tumor development and growth. Biochemical and molecular examination of this hypothesis in vitro will be performed in human patient breast tissue specimens and in several human breast cancer cell systems currently in use in our laboratories. These breast cell systems include immortalized transformed breast epithelial cells grown in monolayers on plastic or on extracellular matrix support (ecm such as Matrigel TM or collagen I) and cultures of human breast stromal cells isolated from breast cancer tissues or normal breast tissues. Traditional culturing of cells on plastic promotes maximal proliferation, while culturing cells on collagen or on Matrigel supports cell maintenance and subsequent differentiation. The specific aims of the research on the various human breast cell systems are (1) to examine the extent of aromatase (CYP19) expression and cyclooxygenase (COX-1 and COX-2) expression in human breast cancer specimens, (2) to examine enzyme expression and the extent of formation of E2 in human breast cancer cell systems, (3) to examine the extent of formation of oxidative metabolites in human breast cancer cell systems, and (4) to determine the biological consequences of estrogen biosyntesis and metabolism in the human breast cell systems.